Mutant/mutation
The mutant lacks expression of asparagine synthetase

Protein (function)
Plasmodium lacks the canonical pathways for amino acid biosynthesis and, therefore, relies mainly on host haemoglobin degradation and extracellular sources to meet its amino acid requirements. Although auxotrophic for most of its amino acids, the parasite genome encodes a few enzymes synthesizing glycine, proline, glutamate, glutamine, aspartate and asparagine, the functional significance of which remains unknown. Of these amino acids, asparagine plays a pivotal role in the parasite life cycle by serving as one of the most abundant amino acids in Plasmodium proteins. The malaria parasite has retained asparagine synthetase (AS) that catalyses the formation of asparagine from aspartate.

Asparagine biosynthesis is catalysed by two evolutionarily independent families of enzymes—AS-A and AS-B, of which AS-A catalyses the amidation of aspartate using ammonia as nitrogen source, whereas AS-B can utilize both glutamine and ammonia though glutamine is preferred under physiological conditions. Sequence analysis of the annotated Plasmodium AS revealed that the parasite enzyme belongs to AS-B, comprising an N-terminal glutamine-hydrolyzing domain representing class-II glutamine amidotransferases/N-terminal aminohydrolases superfamily and a C-terminal domain representing ATP pyrophosphatases/ adenine nucleotide alpha hydrolases superfamily. The ammonia released during glutamine hydrolysis traverses an intramolecular tunnel and reaches the active site of C-terminal domain where it reacts with b-aspartyl-AMP intermediate to form asparagine.

(In the paper evidence is presented that the parasite enzyme possesses AS-B activity)

Phenotype
(Slightly) reduced growth of blood stages in mice. Reduced ookinete, oocyst and sporozoite production. Prolonged prepatent period after injecting mice intravenously with isolated sporozoites (8 days compared to 6 days of wild type sporozoites).

Additional information
By treating mice with L-aparaginase (in order to deplete asparagine) and analysis of development of the mutant parasites evidence is presented that the lack of exogenous asparagine in combination with the absence of asparagine synthetase completely blocks ookinete and liver stage development (and blocks exflagellation of male gametocytes). Results indicate that extracellular asparagine is essential for in vivo liver-stage development.


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